A. Field of the Invention.
The present invention relates to methods of protecting and preserving connective and support tissues by utilizing prostaglandin E.sub.1 (PGE.sub.1) compounds or cyclic adenosine monophosphate (cAMP) agonists or inducers of endogenous cAMP to block collagenase gene expression. Since connective and support tissues, which comprise skin, bone, cartilage, fascia, tendons, ligaments and collagen containing elements that encapsulate organs, have as their major organic constituent the protein collagen, these methods provide new treatment modalities useful in protecting humans and animals against connective and support tissue degradation that occurs in aging, osteoporosis and osteoarthritis.
Several types of collagen exist: skin, bone, fascia, tendons and ligaments are comprised primarily of type I collagen; cartilage is largely comprised of type II collagen. Breakdown or degradation of these tissues requires the enzyme collagenase (EC 3,424.7), this unique neutral metalloproteinase is produced by cells normally present in connective and support tissues. In certain metabolic disorders or inflammatory diseases, increased levels of this enzyme is produced leading to the accelerated destruction or breakdown of connective and support tissues.
Only collagenase is currently known to be capable of the initial cleavage necessary to degrade type I, II and III collagens. Therefore, regulation of collagenase production can be a critical point at which to control connective and support tissue breakdown and thereby prevent the disorders that this breakdown causes. Examples of conditions or diseases caused by accelerated connective and support tissue breakdown include but are not limited to aging, osteoporosis and arthritis. Prior art approaches to preventing accelerated connective and support tissue breakdown have focused on regulating the effect of collagenase or providing replacement tissue. However, none of the prior art methods have been fully effective, universally applicable and risk free.
B. Description of the Prior Art.
In contrast to the methods of the present invention, prior art methods for protecting or preserving connective and support tissue focussed almost exclusively on preventing tissue inflammation. Previously described methods for blocking collagenase have utilized peptide inhibitors of the formed (previously synthesized) collagenase enzyme or agents that generally suppress immune or connective tissue metabolic function or are toxic for the tissues.
At the present time no specific therapies exist to prevent collagen breakdown associated with aging. In the skin the attritional loss of collagen from breakdown and decreased synthesis of new collagen results in a thinning and the loss of structural integrity. The consequence of these changes is a looseness and wrinkling of skin. Physicians have used injections of bovine collagen to temporarily restore skin fullness but this xenograft material is soon resorbed, that is, removed by immune cells. The injection of foreign collagen does not restore tissue strength as it is not incorporated into the normal architecture of the extracellular matrix. The injection of this foreign protein into the skin has been associated with severe immune reactions and has been reported to cause severe autoimmune responses clinically producing collagen vascular diseases that actually accelerate connective and support tissue breakdown. No proven therapies for preserving skin integrity by protecting skin collagen from collagenase-action have been reported.
The loss of the organic (collagen) and inorganic (mineral) matrices of bone characterizes the pathologic process that leads to osteoporosis. Several agents and drugs have been used to prevent the excessive breakdown of bone in order to treat osteoporosis. The efficacy of these treatment modalities has usually been assessed by following the mineral (calcium) content of bone or its loss from the body. Therapies have not been directed at preventing collagen breakdown as the means of protecting and preserving bone.
Calcitonin is a U.S. Food and Drug Administration approved medication for the treatment of low bone (mineral) mass. Calcitonin is a naturally produced hormone which in pharmacologic amounts can transiently inhibit bone resorbing cells (osteoclasts) and thereby block bone breakdown. This hormone acts via cell surface receptors to activate biochemical signals within the osteoclast leading to a decrease in metabolic activity.
Estrogen, the female sex steroid, is an approved medication for the treatment of postmenopausal osteoporosis. While its mechanism of action is not fully understood, it can induce bone cells to synthesize new collagen as well as endogenous growth factors such as insulin-like growth factor-1 (IGF-1) (Ernst, et. al, "Estradiol Effects on proliferation, messenger RNA for collagen and insulin like growth factor-1 and parathyroid hormone stimulated adenylate activity on osteoblastic cells from calvariae and long bone." Endocrinology 125:825-833 (1989)). However, the benefits of estrogen are limited to perimenopausal women and estrogen treatment is associated with increased risks of uterine and breast cancer (Bergkvist, et. al, "The risk of breast cancer after estrogen and estrogen-progestin replacement." New Engl J Med. 321:293-297 (1989)).
Recently, naturally produced substances have been discovered which promote growth and healing of connective and support tissues. Such substances have been termed growth factors. Growth factors, usually proteins, initiate programs of differentiation and/or development within an organism. Although growth factors would appear to be ideal for inducing support and connective tissue repair, such factors are not practically useful as pharmaceutical agents. Growth factors are proteins and like most proteins are not stable and tend to break down upon storage. As proteins, growth factors are not suitable for oral administration since they are digested and destroyed before entering the blood stream. Growth factors cannot be satisfactorily administered as topical ointments. As a result of the lability of growth factors the preferred route of administration is parenteral, that is, injected into the body or blood under medical supervision. Unfortunately, many of the growth factors are species specific and are recognized as foreign by other species. Thus, there is the constant danger of eliciting an immune response. Lastly, growth factors when administered parenterally, do not necessarily target to skin, connective and support tissues and are more likely to affect many different cells throughout the body. With the exception of certain recently described naturally occurring growth factors, there is no disclosure or suggestion in the prior art of pharmaceutically acceptable compounds that can protect and preserve connective and support tissues by preventing collagenase gene expression, the critical enzyme needed for collagen breakdown.
Osteoarthritis is a disease characterized by inflammation and degradation of the cartilage and subchondral (adjacent) bone in joints. The erosion of connective and support tissues is mediated in large part by proteolytic enzymes (including collagenase) released by cells involved in the inflammatory process within the joint. Collagenase is produced by most of the cell types found in inflamed joints including fibroblasts (Goldberg, G. L.; et. al, "Human fibroblast collagenase." J. Biol Chem 261:6600-6605 (1986)), monocytes and macrophages (Campbell, E. J.; et, al, "Monocytes procollagenase and tissue inhibitor metalloproteinases" J. Biol Chem 262 15862-15868 (1987)), endothelial cells (Herron, G. S.; et. al, "Secretion of metalloproteinases by stimulated capillary endothelial cells." J. Biol Chem 261:2810-2813 (1986)), and synoviocytes (Harris, Jr., E. D. "Pathogenesis of rheumatoid arthritis." In Textbook of Rheumatology, pp. 886-914, Kelly, W. N.; et. al, (eds.) (W. B. Saunders, Phil Pa. (1984); and Georgescu, H. I. "HIG-82 an established cell line from rat periarticular soft tissue which retains the `activable` phenotype." In Vitro Cell and Dev. Biol 24:1015-1022 (1988)). Collagenase derived from the synoviocyte is thought to play a central role in mediating collagen and thus the cartilage destruction associated with osteoarthritic disorders (Krane, S. M.; et. al, "Mechanisms of matrix degradation in rheumatoid arthritis." Ann NY Acad Sci 580:340-354 (1990)).
A number of agents and drugs have been employed to prevent and treat the cartilage and bone destruction occurring in joints affected by osteoarthritis. Principle amongst these agents and drugs are steroidal and non-steroidal anti-inflammatory agents and drugs. Steroids include glucocorticoids, hormones produced by the adrenal cortex and the more potent synthetic analogs of these hormones. These agents have many anti-inflammatory activities including the ability to inhibit the proliferation or activity of the immune cells that produce cytokines capable of stimulating collagenase synthesis, release and activation. Unfortunately, anti-inflammatory steroids have a potent catabolic effect on matrix producing cells, therefore they block the synthesis of new matrix components. The consequence of such treatment is the thinning and weakening of connective and support tissues as well as a decreased ability of these tissues to repair themselves.
Non-steroidal anti-inflammatory agents (NSAIDS) such as aspirin, acetaminophen, ibuprofen, flurbiprofen, etc. act by inhibiting the synthesis or action of key "second messengers" such as prostaglandins. These second messengers carry the "inflammatory signal" to effector cells capable of breaking down connective and support tissues (Vane, J. R. "Prostaglandins as mediators of inflammation." In Advances in Prostaglandin and Thromboxane Research, pp. 791-801, B. Samuelsson and R. Paoletti (eds.), Raven Press NY (1976)). In principle, blocking the signal-chain should prevent the synthesis and activation of the effector molecules that are directly responsible for connective and support tissue destruction. Unfortunately, many signal pathways exist and agents such as the NSAIDS are not effective in fully blocking the inflammatory response seen in osteoarthritic joints nor have they been proven to prevent the connective and support tissue destruction that characterized degenerative joint disease such as osteoarthritis. NSAIDS do not act by inhibiting collagenase gene expression, indeed to one skilled in the art, it is evident based on the data presented in this disclosure that blocking PGE.sub.1 with NSAIDS would block the beneficial effects of PGE.sub.1 which is to suppress collagenase gene expression.
Considerable scientific interest has focused on naturally occurring and synthetic molecules which inhibit neutral metalloproteinases. This class of neutral enzymes, of which collagenase is the prime example are thought to be critical for connective and support tissue breakdown. Natural and synthetic inhibitors have been found capable of blocking the activity of endogenously synthesized, active enzyme collagenase. These inhibitors act directly on the formed enzyme to chemically block its activity, these inhibitors do not act at the level of gene expression. Unfortunately, at the present time, no pharmaceutically acceptable or therapeutically effective collagenase inhibitor is available. Furthermore, should such a compound be produced, there is no indication that it could be properly targeted to connective and support tissues. Cartilage for example is essentially devoid of a vascular supply making drug delivery difficult. Many of the potent, naturally occurring metalloproteinase inhibitors are small peptides. As mentioned earlier with regard to "growth factors" such inhibitor peptides would require parenteral administration and are potentially immunogenic which makes the likelihood that they would be acceptable therapeutic agents remote. Up until the present invention there has been no fully acceptable method for inhibiting the expression of collagenase, the critical enzyme that causes connective and support tissue breakdown. With the exception of certain recently described naturally occurring growth factors, there is no disclosure or suggestion in the prior art of a pharmaceutically acceptable compound that can block collagenase gene expression in connective and support tissues and thereby reduce collagenase activity.
Moreover, the prior art appears to teach away from the findings of the present invention. Prostaglandins are hydroxylated fatty acids which can be produced by many tissues in the body usually in response to inflammatory stimuli. Prostaglandins have been characterized as mediators of inflammation and are thought to be involved in the accelerated connective and support tissue breakdown associated with rheumatic disorders. Prostaglandins of the E.sub.2 class, the class most commonly produced by mammalian cells and the class most commonly studied, stimulate collagenase activity in cells derived from connective and support tissues. Thus, in contrast to prior disclosures that prostaglandins of the E.sub.2 class stimulate collagenase activity, one novel aspect of the present invention is that prostaglandins of the E.sub.1 series can actually block collagenase activity and that prostaglandin E.sub.1 and cyclic adenosine monophosphate block collagenase activity by inhibiting collagenase gene expression.